Haplotype-resolved gapless genome and chromosome segment substitution lines facilitate gene identification in wild rice.

The abundant genetic variation harbored by wild rice (Oryza rufipogon) has provided a reservoir of useful genes for rice breeding. However, the genome of wild rice has not yet been comprehensively assessed. Here, we report the haplotype-resolved gapless genome assembly and annotation of wild rice Y476. In addition, we develop two sets of chromosome segment substitution lines (CSSLs) using Y476 as the donor parent and cultivated rice as the recurrent parents. By analyzing the gapless reference genome and CSSL population, we identify 254 QTLs associated with agronomic traits, biotic and abiotic stresses. We clone a receptor-like kinase gene associated with rice blast resistance and confirm its wild rice allele improves rice blast resistance. Collectively, our study provides a haplotype-resolved gapless reference genome and demonstrates a highly efficient platform for gene identification from wild rice.

OsPRMT6a-mediated arginine methylation of OsJAZ1 regulates jasmonate signaling and spikelet development in rice.

Although both protein arginine methylation (PRMT) and jasmonate (JA) signaling are crucial for regulating plant development, the relationship between these processes in the control of spikelet development remains unclear. In this study, we used the CRISPR/Cas9 technology to generate two OsPRMT6a loss-of-function mutants that exhibit various abnormal spikelet structures. Interestingly, we found that OsPRMT6a can methylate arginine residues in JA signal repressors OsJAZ1 and OsJAZ7. We showed that arginine methylation of OsJAZ1 enhances the binding affinity of OsJAZ1 with the JA receptors OsCOI1a and OsCOI1b in the presence of JAs, thereby promoting the ubiquitination of OsJAZ1 by the SCFOsCOI1a/OsCOI1b complex and degradation via the 26S proteasome. This process ultimately releases OsMYC2, a core transcriptional regulator in the JA signaling pathway, to activate or repress JA-responsive genes, thereby maintaining normal plant (spikelet) development. However, in the osprmt6a-1 mutant, reduced arginine methylation of OsJAZ1 impaires the interaction between OsJAZ1 and OsCOI1a/OsCOI1b in the presence of JAs. As a result, OsJAZ1 proteins become more stable, repressing JA responses, thus causing the formation of abnormal spikelet structures. Moreover, we discovered that JA signaling reduces the OsPRMT6a mRNA level in an OsMYC2-dependent manner, thereby establishing a negative feedback loop to balance JA signaling. We further found that OsPRMT6a-mediated arginine methylation of OsJAZ1 likely serves as a switch to tune JA signaling to maintain normal spikelet development under harsh environmental conditions such as high temperatures. Collectively, our study establishes a direct molecular link between arginine methylation and JA signaling in rice.

FLOURY ENDOSPERM24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice.

Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure. Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map-based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant. The flo24 mutant develops more severe floury endosperm when grown under high-temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp-2 to regulate starch biosynthesis and endosperm development in rice. Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.

Rice LIKE EARLY STARVATION1 cooperates with FLOURY ENDOSPERM6 to modulate starch biosynthesis and endosperm development.

In cereal grains, starch is synthesized by the concerted actions of multiple enzymes on the surface of starch granules within the amyloplast. However, little is known about how starch-synthesizing enzymes access starch granules, especially for amylopectin biosynthesis. Here, we show that the rice (Oryza sativa) floury endosperm9 (flo9) mutant is defective in amylopectin biosynthesis, leading to grains exhibiting a floury endosperm with a hollow core. Molecular cloning revealed that FLO9 encodes a plant-specific protein homologous to Arabidopsis (Arabidopsis thaliana) LIKE EARLY STARVATION1 (LESV). Unlike Arabidopsis LESV, which is involved in starch metabolism in leaves, OsLESV is required for starch granule initiation in the endosperm. OsLESV can directly bind to starch by its C-terminal tryptophan (Trp)-rich region. Cellular and biochemical evidence suggests that OsLESV interacts with the starch-binding protein FLO6, and loss-of-function mutations of either gene impair ISOAMYLASE1 (ISA1) targeting to starch granules. Genetically, OsLESV acts synergistically with FLO6 to regulate starch biosynthesis and endosperm development. Together, our results identify OsLESV-FLO6 as a non-enzymatic molecular module responsible for ISA1 localization on starch granules, and present a target gene for use in biotechnology to control starch content and composition in rice endosperm.

A CYP78As-small grain4-coat protein complex â ¡ pathway promotes grain size in rice.

CYP78A, a cytochrome P450 subfamily that includes rice (Oryza sativa L.) BIG GRAIN2 (BG2, CYP78A13) and Arabidopsis thaliana KLUH (KLU, CYP78A5), generate an unknown mobile growth signal (referred to as a CYP78A-derived signal) that increases grain (seed) size. However, the mechanism by which the CYP78A pathway increases grain size remains elusive. Here, we characterized a rice small grain mutant, small grain4 (smg4), with smaller grains than its wild type due to restricted cell expansion and cell proliferation in spikelet hulls. SMG4 encodes a multidrug and toxic compound extrusion (MATE) transporter. Loss of function of SMG4 causes smaller grains while overexpressing SMG4 results in larger grains. SMG4 is mainly localized to endoplasmic reticulum (ER) exit sites (ERESs) and partially localized to the ER and Golgi. Biochemically, SMG4 interacts with coat protein complex Ⅱ (COPⅡ) components (Sar1, Sec23, and Sec24) and CYP78As (BG2, GRAIN LENGTH 3.2 [GL3.2], and BG2-LIKE 1 [BG2L1]). Genetically, SMG4 acts, at least in part, in a common pathway with Sar1 and CYP78As to regulate grain size. In summary, our findings reveal a CYP78As-SMG4-COPⅡ regulatory pathway for grain size in rice, thus providing new insights into the molecular and genetic regulatory mechanism of grain size.

ESCRT-III component OsSNF7.2 modulates leaf rolling by trafficking and endosomal degradation of auxin biosynthetic enzyme OsYUC8 in rice.

The endosomal sorting complex required for transport (ESCRT) is highly conserved in eukaryotic cells and plays an essential role in the biogenesis of multivesicular bodies and cargo degradation to the plant vacuole or lysosomes. Although ESCRT components affect a variety of plant growth and development processes, their impact on leaf development is rarely reported. Here, we found that OsSNF7.2, an ESCRT-III component, controls leaf rolling in rice (Oryza sativa). The Ossnf7.2 mutant rolled leaf 17 (rl17) has adaxially rolled leaves due to the decreased number and size of the bulliform cells. OsSNF7.2 is expressed ubiquitously in all tissues, and its protein is localized in the endosomal compartments. OsSNF7.2 homologs, including OsSNF7, OsSNF7.3, and OsSNF7.4, can physically interact with OsSNF7.2, but their single mutation did not result in leaf rolling. Other ESCRT complex subunits, namely OsVPS20, OsVPS24, and OsBRO1, also interact with OsSNF7.2. Further assays revealed that OsSNF7.2 interacts with OsYUC8 and aids its vacuolar degradation. Both Osyuc8 and rl17 Osyuc8 showed rolled leaves, indicating that OsYUC8 and OsSNF7.2 function in the same pathway, conferring leaf development. This study reveals a new biological function for the ESCRT-III components, and provides new insights into the molecular mechanisms underlying leaf rolling.

A deleterious Sar1c variant in rice inhibits export of seed storage proteins from the endoplasmic reticulum.

KEY MESSAGE: We identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm. Higher plants accumlate large amounts of seed storage proteins (SSPs). However, mechanisms underlying SSP trafficking are largely unknown, especially the ER-Golgi anterograde process. Here, we showed that a rice glutelin precursor accumulation13 (gpa13) mutant exhibited floury endosperm and overaccumulated glutelin precursors, which phenocopied the reported RNAi-Sar1abc line. Molecular cloning revealed that the gpa13 allele encodes a mutated Sar1c (mSar1c) with a deletion of two conserved amino acids Pro134 and Try135. Knockdown or knockout of Sar1c alone caused no obvious phenotype, while overexpression of mSar1c resulted in seedling lethality similar to the gpa13 mutant. Transient expression experiment in tobacco combined with subcellular fractionation experiment in gpa13 demonstrated that the expression of mSar1c affects the subcellular distribution of all Sar1 isoforms and Sec23c. In addition, mSar1c failed to interact with COPII component Sec23. Conversely, mSar1c competed with Sar1a/b/d to interact with guanine nucleotide exchange factor Sec12. Together, we identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm.

A regulatory loop establishes the link between the circadian clock and abscisic acid signaling in rice.

There is a close regulatory relationship between the circadian clock and the abscisic acid (ABA) signaling pathway in regulating many developmental processes and stress responses. However, the exact feedback regulation mechanism between them is still poorly understood. Here, we identified the rice (Oryza sativa) clock component PSEUDO-RESPONSE REGULATOR 95 (OsPRR95) as a transcriptional regulator that accelerates seed germination and seedling growth by inhibiting ABA signaling. We also found that OsPRR95 binds to the ABA receptor gene REGULATORY COMPONENTS OF ABA RECEPTORS10 (OsRCAR10) DNA and inhibits its expression. Genetic analysis showed OsRCAR10 acts downstream of OsPRR95 in mediating ABA responses. In addition, the induction of OsPRR95 by ABA partly required a functional OsRCAR10, and the ABA-responsive element-binding factor ABSCISIC ACID INSENSITIVE5 (OsABI5) bound directly to the promoter of OsPRR95 and activated its expression, thus establishing a regulatory feedback loop between OsPRR95, OsRCAR10, and OsABI5. Taken together, our results demonstrated that the OsRCAR10-OsABI5-OsPRR95 feedback loop modulates ABA signaling to fine-tune seed germination and seedling growth, thus establishing the molecular link between ABA signaling and the circadian clock.

Dwarf and High Tillering1 represses rice tillering through mediating the splicing of D14 pre-mRNA.

Strigolactones (SLs) constitute a class of plant hormones that regulate many aspects of plant development, including repressing tillering in rice (Oryza sativa). However, how SL pathways are regulated is still poorly understood. Here, we describe a rice mutant dwarf and high tillering1 (dht1), which exhibits pleiotropic phenotypes (such as dwarfism and increased tiller numbers) similar to those of mutants defective in SL signaling. We show that DHT1 encodes a monocotyledon-specific hnRNP-like protein that acts as a previously unrecognized intron splicing factor for many precursor mRNAs (pre-mRNAs), including for the SL receptor gene D14. We find that the dht1 (DHT1I232F) mutant protein is impaired in its stability and RNA binding activity, causing defective splicing of D14 pre-mRNA and reduced D14 expression, and consequently leading to the SL signaling-defective phenotypes. Overall, our findings deepen our understanding of the functional diversification of hnRNP-like proteins and establish a connection between posttranscriptional splicing and SL signaling in the regulation of plant development.

OsBSK3 Positively Regulates Grain Length and Weight by Inhibiting the Phosphatase Activity of OsPPKL1.

Brassinosteroids (BRs) are a crucial class of plant hormones that regulate many important agronomic traits in rice (Oryza sativa L.); thus, the BR signaling pathway is a very important tool for breeders to improve the grain yield and quantity of rice. Contrary to the well-established BR signaling pathway in Arabidopsis, there are significant gaps in the rice BR signaling pathway, especially the regulation mechanism from OsBSK3 to OsPPKLs and OsGSKs. In this study, we report how OsBSK3 knockout mutants confer shorter and lighter grains and exhibit a typical BR-insensitive phenotype, suggesting OsBSK3 plays a positive role in BR signaling without genetic redundancy with homologs. Furthermore, OsBSK3 could physically interact with OsPPKL1 and OsGSK3, the downstream components in BR signaling, as a scaffold protein, and inhibit the phosphatase activity of OsPPKL1 on the dephosphorylation of OsGSK3. In addition, the genetic evidence showed OsBSK3 acts upstream of OsPPKL1 in regulating grain length and weight. Our results clarify the role of OsBSK3 and provide new insights into BR-signaling mechanisms, leading to potential new targets for the genetic improvement of rice.

Ectopic Expression of Executor Gene Xa23 Enhances Resistance to Both Bacterial and Fungal Diseases in Rice.

Bacterial blight (BB) and bacterial leaf streak (BLS), caused by phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively, are the most serious bacterial diseases of rice, while blast, caused by Magnaporthe oryzae (M. oryzae), is the most devastating fungal disease in rice. Generating broad-spectrum resistance to these diseases is one of the key approaches for the sustainable production of rice. Executor (E) genes are a unique type of plant resistance (R) genes, which can specifically trap transcription activator-like effectors (TALEs) of pathogens and trigger an intense defense reaction characterized by a hypersensitive response in the host. This strong resistance is a result of programed cell death induced by the E gene expression that is only activated upon the binding of a TALE to the effector-binding element (EBE) located in the E gene promoter during the pathogen infection. Our previous studies revealed that the E gene Xa23 has the broadest and highest resistance to BB. To investigate whether the Xa23-mediated resistance is efficient against Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of BLS, we generated a new version of Xa23, designated as Xa23p1.0, to specifically trap the conserved TALEs from multiple Xoc strains. The results showed that the Xa23p1.0 confers broad resistance against both BB and BLS in rice. Moreover, our further experiment on the Xa23p1.0 transgenic plants firstly demonstrated that the E-gene-mediated defensive reaction is also effective against M. oryzae, the causal agent of the most devastating fungal disease in rice. Our current work provides a new strategy to exploit the full potential of the E-gene-mediated disease resistance in rice.

Rice STOMATAL CYTOKINESIS DEFECTIVE2 regulates cell expansion by affecting vesicular trafficking in rice.

Vesicular trafficking plays critical roles in cell expansion in yeast and mammals, but information linking vesicular trafficking and cell expansion in plants is limited. Here, we isolated and characterized a rice (Oryza sativa) mutant, decreased plant height 1-1 (dph1-1), which exhibited a wide spectrum of developmental phenotypes, including reduced plant height and smaller panicles and grains. Cytological analysis revealed that limited cell expansion was responsible for the dph1-1 mutant phenotype compared to the wild-type. Map-based cloning revealed that DPH1 encodes a plant-specific protein, OsSCD2, which is homologous to Arabidopsis (Arabidopsis thaliana) STOMATAL CYTOKINESIS DEFECTIVE2 (SCD2). Subcellular localization revealed that OsSCD2 is associated with clathrin. Confocal microscopy showed that the dph1-1 mutant has defective endocytosis and post-Golgi trafficking. Biochemical and confocal data indicated that OsSCD2 physically interacts with OsSCD1 and that they are associated with intracellular structures that colocalize with microtubules. Furthermore, we found that cellulose synthesis was affected in the dph1-1 mutant, evidenced by reduced cellulose synthase gene accumulation at the transcript and protein levels, most likely resulting from an impaired localization pattern. Our results suggest that OsSCD2 is involved in clathrin-related vesicular trafficking with an important role in maintaining plant growth in rice.

Genetic characterization and fine mapping of qHMS4 responsible for pollen sterility in hybrids between Oryza sativa L. and Oryza glaberrima Steud.

African cultivated rice (Oryza glaberrima Steud) contains many favorable genes for tolerance to biotic and abiotic stresses and F1 hybrids between Asian cultivated rice (Oryza sativa L.) show strong heterosis. However, the hybrids of two species often exhibit hybrid sterility. Here, we identified a male sterility locus qHMS4 on chromosome 4 (Chr.4), which induces pollen semi-sterility in F1 hybrids of japonica rice variety Dianjingyou1 (DJY1) and a near-isogenic line (NIL) carrying a Chr.4 segment from Oryza glaberrima accession IRGC101854. Cytological observations indicated that non-functional pollen grains produced by the hybrids and lacking starch accumulation abort at the late bicellular stage. Molecular genetic analysis revealed distorted segregation in male gametogenesis carrying qHMS4 allele from DJY1. Fine-mapping of qHMS4 using an F2 population of 22,500 plants delimited qHMS4 to a region of 110-kb on the short arm of Chr.4. Sequence analysis showed that the corresponding sequence region in DJY1 and Oryza glaberrima were 114-kb and 323-kb, respectively, and that the sequence homology was very poor. Gene prediction analysis identified 16 and 46 open reading frames (ORFs) based on the sequences of DJY1 and O. glaberrima, respectively, among which 3 ORFs were shared by both. Future map-based cloning of qHMS4 will help to understand the underlying molecular mechanism of hybrid sterility between the two cultivated rice species. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01306-8.

Post-Golgi trafficking of rice storage proteins requires the small GTPase Rab7 activation complex MON1-CCZ1.

Protein storage vacuoles (PSVs) are unique organelles that accumulate storage proteins in plant seeds. Although morphological evidence points to the existence of multiple PSV-trafficking pathways for storage protein targeting, the molecular mechanisms that regulate these processes remain mostly unknown. Here, we report the functional characterization of the rice (Oryza sativa) glutelin precursor accumulation7 (gpa7) mutant, which over-accumulates 57-kDa glutelin precursors in dry seeds. Cytological and immunocytochemistry studies revealed that the gpa7 mutant exhibits abnormal accumulation of storage prevacuolar compartment-like structures, accompanied by the partial mistargeting of glutelins to the extracellular space. The gpa7 mutant was altered in the CCZ1 locus, which encodes the rice homolog of Arabidopsis (Arabidopsis thaliana) CALCIUM CAFFEINE ZINC SENSITIVITY1a (CCZ1a) and CCZ1b. Biochemical evidence showed that rice CCZ1 interacts with MONENSIN SENSITIVITY1 (MON1) and that these proteins function together as the Rat brain 5 (Rab5) effector and the Rab7 guanine nucleotide exchange factor (GEF). Notably, loss of CCZ1 function promoted the endosomal localization of vacuolar protein sorting-associated protein 9 (VPS9), which is the GEF for Rab5 in plants. Together, our results indicate that the MON1-CCZ1 complex is involved in post-Golgi trafficking of rice storage protein through a Rab5- and Rab7-dependent pathway.

Determinant Factors and Regulatory Systems for Anthocyanin Biosynthesis in Rice Apiculi and Stigmas.

Anthocyanins cause purple, brown or red colors in various tissues of rice plants, but the specific determinant factors and regulatory systems for anthocyanin biosynthesis in almost all tissues remain largely unknown. In the present study, we mapped and isolated two complementary genes, OsC1 encoding a R2R3-MYB transcriptional factor and OsDFR encoding a dihydroflavonol 4-reductase, which are responsible for the purple coloration of apiculi and stigmas in indica cultivar Xieqingzao by the map-based cloning strategy. We also identified two tissue-specific pigmentation genes, OsPa for apiculi and OsPs for stigmas, by phylogenetic analysis of all anthocyanin biosynthesis-associated bHLH transcriptional factors in maize and rice, CRISPR/Cas9 knockout and transcriptional expression analysis. The OsC1, OsPa and OsPs proteins are all localized in the nucleus while the OsDFR protein is localized in the nucleus and cytoplasm, and the OsC1 and OsDFR genes are preferentially strongly expressed in both purple-colored tissues while the OsPa and OsPs genes are preferentially strongly expressed in apiculi and stigmas, respectively. OsC1 specifically interacts with OsPa or OsPs to activate OsDFR and other anthocyanin biosynthesis genes, resulting in purple-colored apiculi or stigmas. OsC1 itself does not produce color but can produce brown apiculi when functioning together with OsPa. Loss of function of OsDFR alone leads to brown apiculi and straw-white stigmas. Genotyping and phenotyping of a panel of 176 rice accessions revealed diverse genotypic combinations of OsC1, OsDFR, OsPa and OsPs that enable accurate prediction of their apiculus and stigma pigmentation phenotypes, thus validating the general applicability of the OsC1-OsDFR-OsPa and OsC1-OsDFR-OsPs models to natural populations. Our findings disclosed the biological functions of OsC1, OsPa and OsPs, and shed light on the specific regulatory systems of anthocyanin biosynthesis in apiculi and stigmas, a further step in understanding the regulatory network of anthocyanin biosynthesis in rice.

ENLARGED STARCH GRAIN1 affects amyloplast development and starch biosynthesis in rice endosperm.

Cereal crops accumulate large amounts of starch which is synthesized and stored in amyloplasts in the form of starch grains (SGs). Despite significant progress in deciphering starch biosynthesis, our understanding of amyloplast development in rice (Oryza sativa) endosperm remains largely unknown. Here, we report a novel rice floury mutant named enlarged starch grain1 (esg1). The mutant has decreased starch content, altered starch physicochemical properties, slower grain-filling rate and reduced 1000-grain weight. A distinctive feature in esg1 endosperm is that SGs are much larger, mainly due to an increased number of starch granules per SG. Spherical and loosely assembled granules, together with those weakly stained SGs may account for decreased starch content in esg1. Map-based cloning revealed that ESG1 encodes a putative permease subunit of a bacterial-type ABC (ATP-binding cassette) lipid transporter. ESG1 is constitutively expressed in various tissues. It encodes a protein localized to the chloroplast and amyloplast membranes. Mutation of ESG1 causes defective galactolipid synthesis. The overall study indicates that ESG1 is a newly identified protein affecting SG development and subsequent starch biosynthesis, which provides novel insights into amyloplast development in rice.

Rice FLOURY ENDOSPERM 18 encodes a pentatricopeptide repeat protein required for 5' processing of mitochondrial nad5 messenger RNA and endosperm development.

Pentatricopeptide repeat (PPR) proteins, composing one of the largest protein families in plants, are involved in RNA binding and regulation of organelle RNA metabolism at the post-transcriptional level. Although several PPR proteins have been implicated in endosperm development in rice (Oryza sativa), the molecular functions of many PPRs remain obscure. Here, we identified a rice endosperm mutant named floury endosperm 18 (flo18) with pleiotropic defects in both reproductive and vegetative development. Map-based cloning and complementation tests showed that FLO18 encodes a mitochondrion-targeted P-type PPR protein with 15 PPR motifs. Mitochondrial function was disrupted in the flo18 mutant, as evidenced by decreased assembly of Complex I in the mitochondrial electron transport chain and altered mitochondrial morphology. Loss of FLO18 function resulted in defective 5'-end processing of mitochondrial nad5 transcripts encoding subunit 5 of nicotinamide adenine dinucleotide hydrogenase. These results suggested that FLO18 is involved in 5'-end processing of nad5 messenger RNA and plays an important role in mitochondrial function and endosperm development.

Transcriptional activation and phosphorylation of OsCNGC9 confer enhanced chilling tolerance in rice.

Low temperature is a major environmental factor that limits plant growth and productivity. Although transient elevation of cytoplasmic calcium has long been recognized as a critical signal for plant cold tolerance, the calcium channels responsible for this process have remained largely elusive. Here we report that OsCNGC9, a cyclic nucleotide-gated channel, positively regulates chilling tolerance by mediating cytoplasmic calcium elevation in rice (Oryza sativa). We showed that the loss-of-function mutant of OsCNGC9 is defective in cold-induced calcium influx and more sensitive to prolonged cold treatment, whereas OsCNGC9 overexpression confers enhanced cold tolerance. Mechanistically, we demonstrated that in response to chilling stress, OsSAPK8, a homolog of Arabidopsis thaliana OST1, phosphorylates and activates OsCNGC9 to trigger Ca2+ influx. Moreover, we found that the transcription of OsCNGC9 is activated by a rice dehydration-responsive element-binding transcription factor, OsDREB1A. Taken together, our results suggest that OsCNGC9 enhances chilling tolerance in rice through regulating cold-induced calcium influx and cytoplasmic calcium elevation.